POS0470 FOSL-2 TRANSCRIPTION FACTOR REGULATES MACROPHAGE POLARIZATION AND PHAGOCYTOSIS IN SYSTEMIC SCLEROSIS
Authors: A. Hukara, T. Tabib, M. Rudnik, O. Distler, P. Blyszczuk, R. Lafyatis, G. Kania
Journal: Annals of the Rheumatic Diseases (2022)

Background Macrophages play a crucial role in the development and progression of systemic sclerosis (SSc). Pathological effects of the AP-1 transcription factor Fos-related antigen 2 (Fosl-2) have been associated with SSc. However, the exact role of Fosl-2 in macrophage function in SSc has not been clarified. Objectives To study macrophage functions in SSc with a focus on Fosl-2 as a potential regulator. Methods Human monocyte-derived macrophages (hMDM) were differentiated from CD14 + blood-derived monocytes from healthy controls and SSc patients. Published data of single cell RNA sequencing (scRNAseq) of human explanted lung tissue from SSc-ILD patients 1 and skin from dcSSc patients 2 was further analyzed using the R package Seurat V2.3.4. Peritoneal macrophages were isolated from Fosl2 overexpressing transgenic (Fosl2tg), Csf1R Cre Fosl2 fl/fl , wild-type (wt) and Fosl2 fl/fl mice. Protein expression was detected by Western Blot. Phagocytic activity in hMDM was detected using pHrodo Red E.coli particles and in vivo phagocytosis of phagocytic dye aggregates in peritoneal macrophages was assessed 4 hours post injection and detected by flow cytometry. Surface marker expression was measured using human or mouse macrophage polarization panels by flow cytometry. Results SSc hMDM (n=18-25) showed increased Fosl-2 protein expression (untreated: p&lt;0.01, LPS stimulated: p&lt;0.01) compared to healthy hMDM (n=11-18). Phenotypical characterization of untreated SSc hMDM (n=17) displayed an increased percentage of CD40 + CD86 + CD206 + PD-L2 + CD163 + cells (p&lt;0.05) compared to healthy hMDM (n=7). Additionally, we found enhanced phagocytic activity in untreated (p&lt;0.01) and LPS stimulated (p&lt;0.05) SSc hMDM (n=29-34) compared to healthy hMDM (n=12-16). To assess the role of Fosl-2 in other macrophage types, we performed scRNAseq analysis of the SSc-ILD lung dataset, which lead to the identification of differentially expressed macrophage polarization ( CD206 ) and phagocytosis-associated genes ( MARCO/C1QA/C1QB/C1QC ) in SPP1 hi macrophages from SSc-ILD patients when comparing FOSL2 hi and FOSL2 null cells. Moreover, independent of FOSL2 expression phagocytosis-associated ARPC1B/ARPC2/ARPC5 genes were upregulated in SPP1 hi , FABP4 hi and FCN1 hi macrophages in SSc-ILD patients (p.adj.≤0.05; log2 ratio≥0.5). Similarly, to the alternatively-activated macrophages in SSc-ILD lungs, scRNAseq of dcSSc skin tissue revealed increased expression of CD204/CD163/CD36 in CCR1 + macrophages. Further, FOSL2 hi CCR1 + skin macrophages showed higher expression of phagolysosome-associated CARO1A and ARL8B genes compared to FOSL2 null cells (p.adj.≤0.05; log2 ratio≥0.5). In the immunofibrotic animal model of SSc, Fosl2tg peritoneal macrophages showed enhanced expression of alternatively-activated CD206 (p=0.0591) and PD-L2 (p&lt;0.05) polarization markers compared to wt cells (n=5-9). Csf1R Cre Fosl2 fl/fl peritoneal macrophages displayed a reduced expression of CD206 (p&lt;0.05) and PD-L2 (p&lt;0.01) markers compared to Fosl2 fl/fl controls (n=5-9). Preliminary data indicated that Fosl2tg MHCII + and CD36 + peritoneal macrophages showed a trend towards elevated phagocytic activity compared to wt cells (n=3). Csf1R Cre Fosl2 fl/fl peritoneal macrophages did not show a significant difference in phagocytic activity (n=3) compared to Fosl2 fl/fl controls. Conclusion For the first time, we showed an increased expression of Fosl-2 and boosted phagocytic activity in SSc hMDM. scRNAseq analyses revealed upregulated phagocytosis-related genes with association to alternatively-activated macrophage polarization in different macrophage clusters in SSc-ILD lungs and dcSSc skin. Moreover, our animal data indicated an involvement of Fosl-2 regulating alternatively-activated macrophage polarization and phagocytosis. Therefore, targeting this alternative/pro-phagocytic phenotype represents an effective tool to counteract disease progression. References [1]Valenzi, E., et al., Ann Rheum Dis, 2019 [2]Xue, D., et al., Arthritis Rheumatol, 2022 Disclosure of Interests Amela Hukara: None declared, Tracy Tabib: None declared, Michal Rudnik: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: Abbvie, Acceleron, Alcimed, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, 4P Science, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Prometheus, Roivant, Sanofi and Topadur, Grant/research support from: Kymera, Mitsubishi Tanabe, Boehringer Ingelheim, Przemyslaw Blyszczuk: None declared, Robert Lafyatis Consultant of: Pfizer, Bristol Myers Squibb, Boehringer-Ingleheim, Formation, Sanofi, Boehringer-Mannheim, Merck and Genentech/Roche, Grant/research support from: Corbus, Formation, Moderna, Regeneron, Pfizer and Kiniksa, Gabriela Kania: None declared.